The properties and functions of the membrane and cortex differ significantly in prophase I oocytes and MII eggs (A). (Note: Throughout this article, the term “oocyte” will be used to refer to the female gamete generically and also for GVI, prophase I oocytes; the term “egg” connotes MII arrest.) Oocytes progress through meiotic maturation, characterized by progression through GVBD and MI, and then arrest at MII. Meiotic maturation in vivo occurs with ovulation and transit of the ovulated egg(s) to the oviduct; meiotic maturation can also occur in vitro, with the culture of prophase I oocytes in medium that supports a decrease in protein kinase A activity and the subsequent increase in CDK1 activity (Mehlmann, 2005
). The MII egg is characterized by distinct domains of membrane and underlying cortex: the microvillar (MV) domain to which sperm bind and fuse, and the amicrovillar (AMV) domain, overlying the meiotic spindle and characterized by an actin-rich cap, contrasting the uniform distribution of actin and microvilli at prophase I stage (A). Exit from MII is triggered by fertilization.
Figure 1. Changes in effective tension during meiotic maturation and fertilization. (A) Schematic diagram illustrating progression of the prophase I germinal vesicle-intact (GVI) oocytes through meiotic maturation, through germinal vesicle breakdown (GVBD) and (more ...)
We hypothesized that the mechanical properties of oocytes would vary through these developmental transitions, given the changes that occur during meiosis and fertilization. Teff
measurements using MPA (B) showed that as oocytes matured from prophase I (GVI) to MII, the effective tension varied about sixfold. GVI oocytes had a mean ± SEM Teff
value of 5.9 ± 0.2 nN/μm (C, Figure S1), making oocytes among the more rigid cell types studied (Hochmuth, 2000
decreased through GVBD and metaphase I, reached a nadir at MII, and then increased after fertilization (C, Figure S1). Interestingly, MII eggs were found to have mechanical polarity, with a nearly threefold difference in Teff
detected between the microvillar and amicrovillar domains (D, Figure S1).
This work includes studies using injections of prophase I oocytes followed by in vitro maturation to MII, so we compared the Teff
levels in MII eggs that had been matured in vivo, with gonadotropin-induced ovulation, to the Teff
levels in eggs that had been matured in vitro. Teff
was not different between the microvillar domains of ovulated and in vitro matured eggs (ovulated, 0.84 ± 0.023 nN/μm [n = 200]; in vitro matured, 0.80 ± 0.018 nN/μm [n = 187]; E, Figure S1). On the other hand, Teff
differed between the amicrovillar domains. Amicrovillar domains of ovulated eggs measured 2.3 ± 0.085 nN/μm (n = 91), and amicrovillar domains of in vitro matured eggs measured 2.0 ± 0.069 nN/μm (n = 84); this difference was statistically significant (p = 0.002) (E, Figure S1). Because the MII spindle is sequestered in the amicrovillar domain, we used anti-tubulin staining to investigate if these differences in effective tension correlated with differences in the morphology of the MII spindle between ovulated eggs and in vitro matured eggs. Examples of these are shown in F; spindles in ovulated eggs tended to be shorter and often with narrower poles, whereas spindles in in vitro matured eggs were longer with broader poles, consistent with other studies using similar in vitro maturation methods (Sanfins et al., 2003
; Barrett and Albertini, 2007
); these references provide extensive characterization of spindle morphology in ovulated and in vitro–matured MII eggs.
The actin cytoskeleton plays a major role in cortical dynamics and tension in other cell types, and we show here that this is true for oocytes as well. Cytochalasin D treatment, which disrupts actin polymers, reduced Teff in GVI oocytes by 95% (B, Figure S2). Cytochalasin D–treated MII eggs were aspirated into the pipette once aspiration pressure was applied, making it impossible to obtain precise Teff values but suggesting that these eggs had extremely low Teff. Because of the sixfold difference in Teff between oocytes and eggs, we hypothesized that actin levels would be higher in GVI oocytes as compared with MII eggs. However, quantitative immunoblot analysis revealed that oocytes and eggs had comparable concentrations of total, polymeric, and soluble actin (C), prompting us to consider additional tension-regulating factors to account for this Teff difference between GVI oocytes and MII eggs.
Myosin-II contractility works with actin to mediate cortical tension in other cell types, and is regulated by phosphorylation of the myosin-II regulatory light chain. We find that the active, phosphorylated form of the myosin-II regulatory light chain (hereafter referred to as pMRLC) is detected in the oocyte/egg and is enriched in the cortex of oocytes and in the amicrovillar domain of eggs (, A–K), colocalized with actin polymers (A) and myosin-II heavy chains IIA and IIB (Maro et al., 1984
; Simerly et al., 1998
). The role of myosin-II in cortical tension and cell shape in mitotic cells has been characterized through manipulation of myosin-II–mediated contractility (Pasternak and Elson, 1985
; Pasternak et al., 1989
; Lucero et al., 2006
; Kunda et al., 2008
); we used these methods to assess myosin-based mechanics in oocytes and eggs. Treatment of cells with concanavalin A (ConA) typically induces a myosin-II–dependent increase in cortical tension (Pasternak and Elson, 1985
; Pasternak et al., 1989
; Kunda et al., 2008
). ConA-treated eggs showed an increase in Teff
in the microvillar domain compared with untreated controls (50%; p < 0.001, Mann-Whitney U-test) and a modest decrease in Teff
of the amicrovillar domain relative to controls (18%; p = 0.14, Mann-Whitney U-test; L, Figure S3). These observations suggest that ConA treatment alters the normal mechanical polarity in MII eggs. The localizations of pMRLC and phosphorylated ERM (addressed below) were not dramatically different between controls and ConA-treated eggs (Figure S3).
Figure 3. Localization of pMRLC and effects of myosin-II manipulation on effective tension. (A–K) Immunofluorescence localization of phosphorylated myosin-II regulatory light chain (pMRLC) in oocytes and eggs. Stages shown are GVI oocyte (A–D) and (more ...)
Inhibition of myosin light-chain kinase (MLCK) activity with ML-7 affects cortical contractility in sea urchin zygotes (Lucero et al., 2006
) and alters certain events of mouse oocyte maturation and egg activation (Matson et al., 2006
; Li et al., 2008a
; Schuh and Ellenberg, 2008
). We show that ML-7-treated oocytes and eggs had a ~50% decrease in Teff
; this decrease in Teff
in MII eggs was observed in both the microvillar and amicrovillar domains (M, Figure S2). These effects of ML-7 treatment suggest a connectedness of the cortical cytoskeleton as well as the fact that low concentrations of myosin II and/or low levels of myosin II activation (i.e., in the microvillar vs. amicrovillar domains, respectively) are sufficient to impact egg mechanics (Zhang and Robinson, 2005
; Kunda et al., 2008
; Reichl et al., 2008
We next examined the ERM protein family. Moesin in Drosophila
and enlazin, the closest ERM relative in Dictyostelium
, contribute to cortical mechanics (Octtaviani et al., 2006
; Kunda et al., 2008
). Interestingly, radixin is very abundantly represented in the mouse oocyte transcriptome (e.g., Evsikov et al., 2006
; Unigene, www.BioGPS.org
), but nothing is known of its function. We show here that mouse eggs express radixin and moesin (, A–C), complementing reports of ezrin expression (Louvet et al., 1996
). Radixin is detectable in lysates of five eggs (125 ng protein) with a band of comparable intensity to that detected in 1 μg of mouse liver lysate (Cii) and is present in the cortex of GVI oocytes and MII eggs (A), in a localization similar to that observed for ezrin (Louvet et al., 1996
). The activity of ERMs is regulated by phosphorylation of a conserved threonine residue in the ERM C-terminal domain that stabilizes a conformation change that allows interactions with ligands, with actin filaments through the C-terminus domain and with the membrane through the FERM (4.1-ezrin-radixin-moesin) domain (Bretscher et al., 2002
; Niggli and Rossy, 2008
; Fehon et al., 2010
). Active pERMs are localized to cortical regions of oocytes and eggs, with enrichment in the microvillar domain of eggs (B). pERM levels decline during meiotic maturation to MII and then increase after fertilization (, D–F), mirroring the changes in Teff
during these developmental transitions (C).
Figure 4. ERMs in mouse eggs. (A and B) Immunofluorescence analysis of radixin (A) and pERM (B) in GVI oocytes (Ai–iv; Bi–iii) and MII eggs (Av–xii and Biv–ix), showing anti-radixin (Aii, vi, and x), anti-pERM (Bii, v, and viii), (more ...)
ERM activity was disrupted through a dominant-negative approach (Skoudy et al., 1999
) by injecting oocytes with cRNA encoding the N-terminal ERM association domain of radixin tagged with the c-myc epitope, hereafter referred to as DN-RDX. Mouse ERM knockout studies show that there is significant functional redundancy between ezrin, radixin, and moesin (Doi et al., 1999
; Kikuchi et al., 2002
; Saotome et al., 2004
), providing strong rationale for a dominant-negative approach to perturb egg ERM function, because mouse eggs express all three ERM family members ( and Louvet et al., 1996
). Staining with an anti-cmyc antibody showed that one-third of eggs injected with DN-RDX cRNA expressed detectable DN-RDX protein. This subset of eggs expressing DN-RDX protein had pERM levels reduced by 84% as determined by anti-pERM staining and analysis of the ratio of signal intensity in the cortex to the total signal intensity (Icortex
; this was a way to normalize between individual eggs; B). We also assessed cortical actin as a control and found that there was no difference in the Icortex
of actin in uninjected, water-injected, or cRNA-injected eggs with decreased pERM (B).
Figure 5. Perturbation of ERM action in eggs. (A) Analysis of DN-RDX-cmyc and pERM by immunofluorescence in uninjected (i–iii), water-injected (iv–vi), and DN-RDX-cRNA-injected eggs (vii–ix). DN-RDX-cmyc protein was detected in ~33% (more ...)
Effective tension levels were assessed in eggs injected with the DN-RDX cRNA and in control eggs (uninjected, water-injected). The entire population of DN-RDX–injected eggs had a mean Teff of 0.75 ± 0.048 nN/μm (Figure S2; p = 0.16; [Mann Whitney U-test] when these eggs were compared with the entire population of in vitro matured MII eggs [0.80 ± 0.018 nN/μm]), but this distribution in Teff levels in DN-RDX–injected eggs is likely due to the fact that only about a third of these eggs express detectable DN-RDX protein. We were able to recover some eggs after the Teff measurements and examine these for DN-RDX expression and reduction of pERM. The cRNA-injected eggs that had detectable DN-RDX protein, and reduced pERM levels had a mean Teff 0.28 ± 0.085 nN/μM, which was significantly different from control eggs (C, Figure S2). We also observed that a small number of DN-RDX–injected eggs deformed as soon as aspiration pressure was applied, similar to the cytochalasin D–treated eggs, suggesting that these eggs had extremely low Teff.
We next sought to examine the biological effects associated with disrupted effective tension in eggs. Meiotic maturation and emission of the first polar body in DN-RDX cRNA-injected oocytes to MII appeared to occur normally. To determine if Teff
disruption affected function of MII egg function and the second meiotic division, we performed in vitro fertilization with control and DN-RDX–injected eggs as well as with cytochalasin D– and ML-7–treated eggs. These eggs could be fertilized but developed significant spindle abnormalities during exit from MII. In normal completion of meiosis II in control eggs (solvent [DMSO]-treated, uninjected, water-injected; , A–C, J–M, and Q–T), the meiotic spindle elongates in anaphase II and then undergoes spindle rotation from its orientation parallel to the membrane. This rotation produces the second polar body, with the polar body developing from one of the chromatin-containing protrusions that forms before spindle rotation; the other protrusion resolves (). Defects in second polar body emission and spindle rotation were observed in cytochalasin D– and ML-7–treated eggs, in agreement with previous studies (Maro et al., 1984
; Matson et al., 2006
); interestingly, spindle rotation failed in DN-RDX–expressing eggs but the spindle abnormalities differed from those in cytochalasin D– and ML-7–treated eggs. (Note: Polar body emission and spindle rotation appeared to be normal in ConA-treated eggs; see Figure S3 for details. This lack of an effect on polar body emission could be due to having to introduce ConA treatment after zygote creation, and/or due to the ConA-induced change in tension being insufficient to induce significant abnormalities during polar body emission.) In cytochalasin D–treated eggs, ML-7–treated eggs, and DN-RDX–expressing eggs, spindle elongation occurred but spindle rotation failed. The cytochalasin D–treated and ML-7–treated eggs arrested at this anaphase II-like stage and no polar body was formed, with only very modest protrusions over the chromatin (, D–I; Maro et al., 1984
; Matson et al., 2006
). Fertilized DN-RDX–expressing eggs developed spindles that were distorted and curved; a pair of extended polar body-like structures was associated with the chromatin at the ends of these distorted spindles (, N–P). These PB-like structures did not resolve, still persisting at 4 h after inseminations (, U-BB).
Figure 6. Spindle defects upon exit from metaphase II arrest with actin, myosin-II, or ERM disruption. Eggs were inseminated for 1.5 h (A–P) or 4 h (Q-BB). Control fertilized eggs (A–C, J–M, and Q–T) show normal spindle rotation (more ...)